The present invention relates to improved variants of soluble pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenases (s-GDH), to genes encoding mutated s-GDH, to mutant proteins of s-GDH with improved substrate specificity for glucose, and to different applications of these s-GDH variants, particularly for determining concentrations of sugar, especially of glucose in a sample.
The International Union of Biochemistry and Molecular Biology (IUBMB) has changed the designation of PQQ-dependent glucose dehydrogenase from EC 1.1.99.17 to EC 1.1.5.2. Accordingly, hereafter, the designation of PQQ-dependent glucose dehydrogenase is recited as EC 1.1.5.2.
Two types of PQQ-dependent glucose dehydrogenase (EC 1.1.5.2) have been characterized: One is membrane-bound (m-GDH), the other is soluble (s-GDH). Both types do not share any significant sequence homology (Cleton-Jansen, A. M., et al., Mol Gen Genet 217 (1989) 430-6; Cleton-Jansen, A. M., et al., Antonie Van Leeuwenhoek 56 (1989) 73-9; Oubrie, A., et al., Proc Natl Acad Sci USA 96 (1999) 11787-91). They are also different regarding both their kinetic as well as their immunological properties (Matsushita, K., et al., Bioscience Biotechnology & Biochemistry 59 (1995) 1548-1555).
Quinoproteins use quinone as cofactor to oxidize alcohols, amines and aldoses to their corresponding lactones, aldehydes and aldolic acids (Duine, J. A. Energy generation and the glucose dehydrogenase pathway in Acinetobacter in “The Biology of Acinetobacter” (1991) 295-312, New York, Plenum Press; Duine, J. A., Eur J Biochem 200 (1991) 271-84, Davidson, V. L.—in “Principles and applications of quinoproteins” (1993) the whole book, New York, Marcel Dekker; Anthony, C., Biochem J 320 (1996) 697-711; Anthony, C. and Ghosh, M., Current Science 72 (1997) 716-727; Anthony, C., Biochem Soc Trans 26 (1998) 413-7; Anthony, C. and Ghosh, M., Prog Biophys Mol Biol 69 (1998) 1-21). Among quinoproteins, those containing the noncovalently bound cofactor 2,7,9-tricarboxy-1H-pyrrolo [2,3-f]quinoline-4,5-dione (PQQ) constitute the largest sub-group (Duine 1991, supra). All bacterial glucose dehydrogenases known so far belong to this sub-group with PQQ as the prosthetic group (Anthony and Ghosh 1997, supra, Goodwin and Anthony 1998, supra).
In bacteria, there are two completely different types of PQQ-dependent glucose dehydrogenases (EC1.1.5.2): the soluble type (s-GDH) and the membrane-bound type (m-GDH) (Duine et al., 1982; Matsushita et al., 1989a,b). The m-GDHs are widespread in Gram-negative bacteria, s-GDHs, however, have been found only in the periplasmic space of Acinetobacter strains, like A. calcoaceticus (Duine, 1991a; Cleton-Jansen et al., 1988; Matsushita and Adachi, 1993) and A. baumannii (JP 11243949).
Through searching sequence databases, two sequences homologous to the full-length A. calcoaceticus s-GDH have been identified in E. coli K-12 and Synechocystis sp. Additionally, two incomplete sequences homologous to A. calcoaceticus s-GDH were also found in the genome of P. aeruginosa and Bordetella pertussis (Oubrie et al. 1999a), respectively. The deduced amino acid sequences of these four uncharacterized proteins are closely related to A. calcoaceticus s-GDH with many residues in the putative active site absolutely conserved. These homologous proteins are likely to have a similar structure and to catalyze similar PQQ-dependent reactions (Oubrie et al., 1999a).
Bacterial s-GDHs and m-GDHs have been found to possess quite different sequences and different substrate specificity. For example, A. calcoaceticus contains two different PQQ-dependent glucose dehydrogenases, one m-GDH which is active in vivo, and the other designated s-GDH for which only in vitro activity can be shown. Cleton-Jansen et al., (1988; 1989a,b) cloned the genes coding for the two GDH enzymes and determined the DNA sequences of both these GDH genes. There is no obvious homology between m-GDH and s-GDH corroborating the fact that m-GDH and s-GDH represent two completely different molecules.
The gene of s-GDH from A. calcoaceticus has been cloned in E. coli behind a leader sequence and a strong promoter. After being synthesized in the cell, the s-GDH is translocated through the cytoplasmic membrane into the periplasmic space (Duine, J. A. Energy generation and the glucose dehydrogenase pathway in Acinetobacter in “The Biology of Acinetobacter” (1991) 295-312, New York, Plenum Press, Matsushita, K. and Adachi, O. Bacterial quinoproteins glucose dehydrogenase and alcohol dehydrogenase in “Principles and applications of Quinoproteins” (1993) 47-63, New York, Marcel Dekker). Like the native s-GDH from A. calcoaceticus, s-GDH expressed in E. coli is also a homodimer, with one PQQ molecule and three calcium ions per monomer (Dokter et al., 1986 supra,1987 supra 1988 supra; Olsthoorn, A. and J. Duine, J. A., Arch Biochem Biophys 336 (1996) 42-8; Oubrie, A., et al., J Mol Biol 289 (1999) 319-33, Oubrie, A., et al., Proc Natl Acad Sci USA 96 (1999) 11787-91, Oubrie, A., et al., Embo J 18 (1999) 5187-94). s-GDH oxidizes a wide range of mono- and disaccharides to the corresponding ketones which further hydrolyze to the aldonic acids, and it is also able to donate electrons to PMS (phenazine metosulfate), DCPIP (2,6-dichlorophenolindophenol), WB (Wurster's blue) and short-chain ubiquinones such as ubiquinone Q1 and ubiquinone Q2 (Matsushita, K., et al., Biochemistry 28 (1989) 6276-80; Matsushita, K., et al., Antonie Van Leeuwenhoek 56 (1989) 63-72), several artificial electron acceptors such as N-methylphenazonium methyl sulfate (Olsthoom, A. J. and Duine, J. A., Arch Biochem Biophys 336 (1996) 42-8; Olsthoorn, A. J. and Duine, J. A., Biochemistry 37 (1998) 13854-61) and electroconducting polymers (Ye, L., et al., Anal. Chem. 65 (1993) 238-41).
In view of s-GDH's high specific activity towards glucose (Olsthoom, A. J. and Duine, J. A., Arch Biochem Biophys 336 (1996) 42-8) and its broad artificial electron acceptor specificity, the enzyme is well suited for analytical applications, particularly for being used in (bio-)sensor or test strips for glucose determination in diagnostic applications (Kaufmann et al., 1997 supra).
Glucose oxidation can be catalyzed by at least three quite distinct groups of enzymes, i.e., by NAD-dependent, dye-linked glucose dehydrogenases, by flavoprotein glucose oxidase or by quinoprotein GDHs (Duine 1995). A rather slow autooxidation of reduced s-GDH has been observed, demonstrating that oxygen is a very poor electron acceptor for s-GDH (Olsthoorn and Duine 1996). s-GDH can efficiently donate electrons to PMS, DCPIP, WB and short-chain ubiquinones such as Q1 and Q2, but it can not efficiently donate electrons directly to oxygen.
Traditional test strips and sensors for monitoring glucose level in blood, serum and urine e. g. from diabetic patients use glucose oxidase. However, since glucose oxidase transfers its electrons to oxygen, it is known that oxygen may have a negative impact on glucose measurements which are based on this enzyme. The major advantage of PQQ-dependent glucose dehydrogenases is their independence from oxygen. This important feature is e.g., discussed in U.S. Pat. No. 6,103,509, in which some features of membrane-bound GDH have been investigated.
An important contribution to the field has been the use of s-GDH together with appropriate substrates. Assay methods and test strip devices based on s-GDH are disclosed in detail in U.S. Pat. No. 5,484,708. This patent also contains detailed information on the set-up of assays and the production of s-GDH-based test strips for measurement of glucose. The methods described there as well in the cited documents are herewith included by reference.
Other patents or applications relating to the field and comprising specific information on various modes of applications for enzymes with glucose dehydrogenase activity are U.S. Pat. No. 5,997,817; U.S. Pat. No. 6,057,120; EP 620 283; and JP 11-243949-A.
A commercial system which utilizes s-GDH and an indicator that produces a color change when the reaction occurs (Kaufmann et al. 1997) is the Glucotrend® system distributed by Roche Diagnostics GmbH.
Despite the above discussed important advantages there also is a major inherent problem of s-GDH. s-GDH has rather a broad substrate spectrum as compared to m-GDH. That is, s-GDH oxidizes not only glucose but also several other sugars including maltose, galactose, lactose, mannose, xylose and ribose (Dokter et al. 1986a). The reactivity towards sugars other than glucose may in certain cases impair the accuracy of determining blood glucose levels, in some diabetic patients. In particular patients on peritoneal dialysis treated with icodextrin (a glucose polymer) may contain in their body fluids, e.g., in blood, high levels of other sugars, especially maltose (Wens, R., et al., Perit Dial Int 18 (1998) 603-9).
Therefore clinical samples, as e.g. obtained from diabetic patients, especially from patients with renal complications and especially from patients under dialysis may contain significant levels of other sugars, especially maltose. Glucose determinations in samples obtained from such critical patients may be impaired by maltose.
There are scarce reports in the literature on attempts to produce modified PQQ-dependent s-GDHs which exhibit altered substrate specificity. Due to a negative outcome most of these efforts have not been published. Igarashi, S., et al., (1999) report that introducing a point mutation at position Glu277 leads to mutants with altered substrate specificity profile. However, none of these mutants, lead to an at least two-fold increased improved specificity for glucose as e.g., compared to xylose, galactose or maltose.
It can be summarized that the attempts known in the art aiming at improvements of properties of s-GDH, especially its specificity towards glucose, have not been successful to the extend required for accurate monitoring of glucose levels in patients having also high levels of sugars other than glucose.
A great demand and clinical need therefore exists for mutant forms of s-GDH which feature an improved specificity for glucose as substrate.
It was the task of the present invention to provide new mutants or variants of s-GDH with significantly improved substrate specificity for glucose as compared to other selected sugar molecules, e.g., like galactose or maltose.
Surprisingly it has been found that it is possible to significantly improve the substrate specificity of s-GDH for glucose, as compared to other sugars, and to at least partially overcome the above described problems known in the art.
The substrate specificity for glucose as compared to other selected sugar molecules has been significantly improved by providing mutant s-GHD according to the present invention as described herein below and in the appending claims. Due to the improved substrate specificity of the new forms of s-GDH, significant technical progress for glucose determinations in various fields of applications is possible.